1 Loading in Data sets + Library packages.

## 
## The downloaded binary packages are in
##  /var/folders/7n/x74qctp91rng390gx0z9hmd80000gn/T//RtmpmqpUvw/downloaded_packages

2 Loading Dataset

options(future.globals.maxSize = 74 * 1024^3) # 55 GB
getOption("future.globals.maxSize") #59055800320
## [1] 79456894976
load(here("jk_code", "SO4_SUB_NEW_analysis.rds"))
Idents(SO4) <- "subclass2_MD"

SO4$sample <- factor(SO4$sample, levels = c("SO1", "SO4", "SO3", "SO2"))

VlnPlot(SO4,"S100g",split.by = "treatment",group.by = "sample")
## The default behaviour of split.by has changed.
## Separate violin plots are now plotted side-by-side.
## To restore the old behaviour of a single split violin,
## set split.plot = TRUE.
##       
## This message will be shown once per session.
## Warning: The `slot` argument of `FetchData()` is deprecated as of SeuratObject 5.0.0.
## ℹ Please use the `layer` argument instead.
## ℹ The deprecated feature was likely used in the Seurat package.
##   Please report the issue at <https://github.com/satijalab/seurat/issues>.
## This warning is displayed once every 8 hours.
## Call `lifecycle::last_lifecycle_warnings()` to see where this warning was
## generated.
## Warning: `PackageCheck()` was deprecated in SeuratObject 5.0.0.
## ℹ Please use `rlang::check_installed()` instead.
## ℹ The deprecated feature was likely used in the Seurat package.
##   Please report the issue at <https://github.com/satijalab/seurat/issues>.
## This warning is displayed once every 8 hours.
## Call `lifecycle::last_lifecycle_warnings()` to see where this warning was
## generated.

# Macula Densa Markers , Nos1, Avpr1a, Slc12a1, Ptgs2, and Slc9a2

VlnPlot(SO4, c("Nos1","Avpr1a","Slc12a1","Ptgs2","Slc9a2","Pappa2"),split.by = "treatment",group.by = "treatment")

DimPlot(SO4)

SO4 <- SO4[
  !grepl("^(mt-|Rp|Gm)|Rik$", rownames(SO4)),
  ,
  drop = FALSE
]
## Warning: The following arguments are not used: drop
DimPlot(SO4,group.by = "treatment",split.by = "treatment")

#compare each type 1 control to type 1 low_treatment

3 Markers of control vs low_salt up/down regulation

Upregulated = low salt, positive, activated by low_salt Downregulated = control, negative, supressed of low_salt

VlnPlot(SO4, features = "Pappa2", group.by = "treatment", split.by = "subclass_MD")

DimPlot(SO4, reduction = "umap", split.by = "treatment", label = TRUE)

Idents(SO4) <- "treatment"

type_markers <- FindMarkers(
  object = SO4,
  ident.1 = "low_salt",   
  ident.2 = "control",     
  group.by = "treatment",
  min.pct = 0.1,           
  logfc.threshold = 0.1,  
  only.pos = FALSE         
)
## Warning: The `slot` argument of `GetAssayData()` is deprecated as of SeuratObject 5.0.0.
## ℹ Please use the `layer` argument instead.
## ℹ The deprecated feature was likely used in the Seurat package.
##   Please report the issue at <https://github.com/satijalab/seurat/issues>.
## This warning is displayed once every 8 hours.
## Call `lifecycle::last_lifecycle_warnings()` to see where this warning was
## generated.
## For a (much!) faster implementation of the Wilcoxon Rank Sum Test,
## (default method for FindMarkers) please install the presto package
## --------------------------------------------
## install.packages('devtools')
## devtools::install_github('immunogenomics/presto')
## --------------------------------------------
## After installation of presto, Seurat will automatically use the more 
## efficient implementation (no further action necessary).
## This message will be shown once per session
 type_markers$gene <- rownames(type_markers)
 
type_markers <-type_markers %>% arrange(desc(avg_log2FC))

3.1 Pathway Upregulated = Low salt

df<- type_markers 


df2 <- df %>% filter(p_val_adj < 0.05)


DEG_list <- df2

markers1up <- DEG_list %>% rownames_to_column(var="SYMBOL")


ENTREZ_list <- bitr(
  geneID = rownames(DEG_list),
  fromType = "SYMBOL",
  toType = "ENTREZID",
  OrgDb = org.Mm.eg.db
)
## 'select()' returned 1:1 mapping between keys and columns
## Warning in bitr(geneID = rownames(DEG_list), fromType = "SYMBOL", toType =
## "ENTREZID", : 3.19% of input gene IDs are fail to map...
markers1up <-  ENTREZ_list %>% inner_join(markers1up, by = "SYMBOL")

markers1up <-  markers1up %>% dplyr::filter(p_val_adj < 0.05)
#head(markers, n = 50)

pos.markers1up <-  markers1up %>% dplyr::filter(avg_log2FC > 0.3) %>%  arrange(desc(abs(avg_log2FC))) 
#change around avg log value 

pos.ranks1up <- pos.markers1up$ENTREZID[abs(pos.markers1up$avg_log2FC) > 0]
#head(pos.ranks)

pos_go1up <- enrichGO(gene = pos.ranks1up,           #a vector of entrez gene id
                   OrgDb = "org.Mm.eg.db",    
                   ont = "BP",
                   readable = TRUE)              #whether mapping gene ID to gene Name

pos_go1up
## #
## # over-representation test
## #
## #...@organism     Mus musculus 
## #...@ontology     BP 
## #...@keytype      ENTREZID 
## #...@gene     chr [1:196] "239435" "16010" "19283" "23850" "19225" "66815" "70337" ...
## #...pvalues adjusted by 'BH' with cutoff <0.05 
## #...260 enriched terms found
## 'data.frame':    260 obs. of  12 variables:
##  $ ID            : chr  "GO:0061077" "GO:0007179" "GO:0010563" "GO:0045936" ...
##  $ Description   : chr  "chaperone-mediated protein folding" "transforming growth factor beta receptor signaling pathway" "negative regulation of phosphorus metabolic process" "negative regulation of phosphate metabolic process" ...
##  $ GeneRatio     : chr  "8/189" "11/189" "14/189" "14/189" ...
##  $ BgRatio       : chr  "69/28928" "218/28928" "388/28928" "388/28928" ...
##  $ RichFactor    : num  0.1159 0.0505 0.0361 0.0361 0.0427 ...
##  $ FoldEnrichment: num  17.75 7.72 5.52 5.52 6.54 ...
##  $ zScore        : num  11.29 8.08 7.27 7.27 7.56 ...
##  $ pvalue        : num  1.70e-08 2.19e-07 3.01e-07 3.01e-07 3.69e-07 ...
##  $ p.adjust      : num  5.17e-05 2.08e-04 2.08e-04 2.08e-04 2.08e-04 ...
##  $ qvalue        : num  3.95e-05 1.59e-04 1.59e-04 1.59e-04 1.59e-04 ...
##  $ geneID        : chr  "Hspb1/Hspa1a/Hspa1b/Hspa8/Hspa5/Clu/Fkbp5/Dnajb1" "Fos/Cav2/Cdkn1c/Bmp2/Bambi/Spry1/Jun/Hspa5/Cited1/Fut8/Tsc22d1" "Hspb1/Dusp1/Cdkn1c/Bmp2/Aldob/Rgs2/Cdk5rap1/Spry1/Ier3/Jun/Sh3bp5/Gstp1/Dynll1/Irf1" "Hspb1/Dusp1/Cdkn1c/Bmp2/Aldob/Rgs2/Cdk5rap1/Spry1/Ier3/Jun/Sh3bp5/Gstp1/Dynll1/Irf1" ...
##  $ Count         : int  8 11 14 14 12 5 12 13 10 12 ...
## #...Citation
## G Yu. Thirteen years of clusterProfiler. The Innovation. 2024, 5(6):100722
 chart1up <- dotplot(pos_go1up) +
    ggtitle("upregulated lowsalt") +
    theme_classic() + 
    theme(
        plot.title = element_text(hjust = 0.5),
        legend.position = "left",
        axis.text.y = element_text(hjust = 0, size = 10)) +
    scale_y_discrete(position = "right", 
                     labels = function(x) str_wrap(x, width = 25))  # Wrap y-axis labels to 2 lines
## Scale for y is already present.
## Adding another scale for y, which will replace the existing scale.
chart1up

### Upregulation Gene View

pathway_type1up <- strsplit(pos_go1up$geneID, "/")

names(pathway_type1up) <- pos_go1up$Description 

pathway_type1up
## $`chaperone-mediated protein folding`
## [1] "Hspb1"  "Hspa1a" "Hspa1b" "Hspa8"  "Hspa5"  "Clu"    "Fkbp5"  "Dnajb1"
## 
## $`transforming growth factor beta receptor signaling pathway`
##  [1] "Fos"     "Cav2"    "Cdkn1c"  "Bmp2"    "Bambi"   "Spry1"   "Jun"    
##  [8] "Hspa5"   "Cited1"  "Fut8"    "Tsc22d1"
## 
## $`negative regulation of phosphorus metabolic process`
##  [1] "Hspb1"    "Dusp1"    "Cdkn1c"   "Bmp2"     "Aldob"    "Rgs2"    
##  [7] "Cdk5rap1" "Spry1"    "Ier3"     "Jun"      "Sh3bp5"   "Gstp1"   
## [13] "Dynll1"   "Irf1"    
## 
## $`negative regulation of phosphate metabolic process`
##  [1] "Hspb1"    "Dusp1"    "Cdkn1c"   "Bmp2"     "Aldob"    "Rgs2"    
##  [7] "Cdk5rap1" "Spry1"    "Ier3"     "Jun"      "Sh3bp5"   "Gstp1"   
## [13] "Dynll1"   "Irf1"    
## 
## $`cellular response to transforming growth factor beta stimulus`
##  [1] "Fos"     "Cav2"    "Cdkn1c"  "Bmp2"    "Bambi"   "Wnt10a"  "Spry1"  
##  [8] "Jun"     "Hspa5"   "Cited1"  "Fut8"    "Tsc22d1"
## 
## $`protein refolding`
## [1] "Hspb1"  "Hspa1a" "Hspa1b" "Hspa8"  "Hspa5" 
## 
## $`response to transforming growth factor beta`
##  [1] "Fos"     "Cav2"    "Cdkn1c"  "Bmp2"    "Bambi"   "Wnt10a"  "Spry1"  
##  [8] "Jun"     "Hspa5"   "Cited1"  "Fut8"    "Tsc22d1"
## 
## $`transforming growth factor beta receptor superfamily signaling pathway`
##  [1] "Fos"     "Cav2"    "Cdkn1c"  "Bmp2"    "Bambi"   "Spry1"   "Hfe"    
##  [8] "Bmper"   "Jun"     "Hspa5"   "Cited1"  "Fut8"    "Tsc22d1"
## 
## $`negative regulation of transferase activity`
##  [1] "Hspb1"    "Dusp1"    "Cdkn1c"   "Bmp2"     "Rgs2"     "Cdk5rap1"
##  [7] "Spry1"    "Zfp36"    "Sh3bp5"   "Gstp1"   
## 
## $`negative regulation of cell adhesion`
##  [1] "Ptprz1" "Hspb1"  "Dusp1"  "Bcl6"   "Bmp2"   "Hfe"    "Arg2"   "Gstp1" 
##  [9] "Irf1"   "Acer2"  "Pik3r1" "Adam15"
## 
## $`chaperone cofactor-dependent protein refolding`
## [1] "Hspa1a" "Hspa1b" "Hspa8"  "Hspa5"  "Dnajb1"
## 
## $`negative regulation of phosphorylation`
##  [1] "Hspb1"    "Dusp1"    "Cdkn1c"   "Bmp2"     "Rgs2"     "Cdk5rap1"
##  [7] "Spry1"    "Jun"      "Sh3bp5"   "Gstp1"    "Dynll1"   "Irf1"    
## 
## $`negative regulation of protein kinase activity`
## [1] "Hspb1"    "Dusp1"    "Cdkn1c"   "Bmp2"     "Rgs2"     "Cdk5rap1" "Spry1"   
## [8] "Sh3bp5"   "Gstp1"   
## 
## $`cell surface receptor protein serine/threonine kinase signaling pathway`
##  [1] "Fos"     "Cav2"    "Cdkn1c"  "Bmp2"    "Bambi"   "Spry1"   "Hfe"    
##  [8] "Bmper"   "Jun"     "Hspa5"   "Cited1"  "Fut8"    "Tsc22d1"
## 
## $`response to heat`
## [1] "Ptgs2"  "Hspb1"  "Hspa1a" "Hspa1b" "Hspa8"  "Nos1"   "Dnajb1"
## 
## $`negative regulation of protein modification process`
##  [1] "Hspb1"    "Dusp1"    "Cdkn1c"   "Bmp2"     "Rgs2"     "Cdk5rap1"
##  [7] "Hspa1b"   "Spry1"    "Jun"      "Sh3bp5"   "Bex2"     "Gstp1"   
## [13] "Irf1"    
## 
## $`negative regulation of kinase activity`
## [1] "Hspb1"    "Dusp1"    "Cdkn1c"   "Bmp2"     "Rgs2"     "Cdk5rap1" "Spry1"   
## [8] "Sh3bp5"   "Gstp1"   
## 
## $`negative regulation of protein phosphorylation`
##  [1] "Hspb1"    "Dusp1"    "Cdkn1c"   "Bmp2"     "Rgs2"     "Cdk5rap1"
##  [7] "Spry1"    "Jun"      "Sh3bp5"   "Gstp1"    "Irf1"    
## 
## $`response to metal ion`
##  [1] "Ptgs2"   "Fos"     "Junb"    "Chp2"    "Aldob"   "Hfe"     "Hspa8"  
##  [8] "Jun"     "Trpc1"   "Pla2g4a" "Slc41a1" "Kcnc2"  
## 
## $`regulation of inclusion body assembly`
## [1] "Hspa1b" "Hap1"   "Clu"    "Dnajb1"
## 
## $`ureteric bud development`
## [1] "Bmp2"   "Spry1"  "Bmper"  "Arg2"   "Cited1" "Robo2"  "Hoxb7" 
## 
## $`'de novo' post-translational protein folding`
## [1] "Hspa1a" "Hspa1b" "Hspa8"  "Hspa5"  "Dnajb1"
## 
## $`mesonephric epithelium development`
## [1] "Bmp2"   "Spry1"  "Bmper"  "Arg2"   "Cited1" "Robo2"  "Hoxb7" 
## 
## $`mesonephric tubule development`
## [1] "Bmp2"   "Spry1"  "Bmper"  "Arg2"   "Cited1" "Robo2"  "Hoxb7" 
## 
## $`'de novo' protein folding`
## [1] "Hspa1a" "Hspa1b" "Hspa8"  "Hspa5"  "Dnajb1"
## 
## $`mesonephros development`
## [1] "Bmp2"   "Spry1"  "Bmper"  "Arg2"   "Cited1" "Robo2"  "Hoxb7" 
## 
## $`positive regulation of cell projection organization`
##  [1] "Ptprz1"  "Ache"    "Hspb1"   "Rgs2"    "Scarf1"  "Lcn2"    "Hap1"   
##  [8] "Dynlt1f" "Dynll1"  "Hspa5"   "Robo2"   "Cx3cl1"  "Pik3r1" 
## 
## $`negative regulation of neuron apoptotic process`
## [1] "Ptprz1" "Btg2"   "Lcn2"   "Jun"    "Cited1" "Tox3"   "Clu"    "Cx3cl1"
## [9] "Nos1"  
## 
## $`kidney epithelium development`
## [1] "Bmp2"   "Spry1"  "Bmper"  "Arg2"   "Cited1" "Robo2"  "Hoxb7"  "Irx1"  
## 
## $`calcium-independent cell-cell adhesion via plasma membrane cell-adhesion molecules`
## [1] "Bmp2"   "Cldn4"  "Cx3cl1" "Cldn3" 
## 
## $`response to temperature stimulus`
## [1] "Ptgs2"  "Hspb1"  "Hspa1a" "Hspa1b" "Hspa8"  "Rbm3"   "Nos1"   "Dnajb1"
## 
## $`regulation of epithelial cell differentiation`
## [1] "Cdkn1c" "Sfn"    "Etv4"   "Bmp2"   "F11r"   "Spry1"  "Zfp36"  "Cited1"
## 
## $`negative regulation of MAP kinase activity`
## [1] "Dusp1" "Bmp2"  "Rgs2"  "Spry1" "Gstp1"
## 
## $regeneration
## [1] "Mcub"   "Mymx"   "Scarf1" "Hfe"    "Cspg5"  "Jun"    "Gstp1"  "Cpq"   
## [9] "Adam15"
## 
## $`protein folding`
## [1] "Hspb1"  "Hspa1a" "Hspa1b" "Hspa8"  "Hspa5"  "Clu"    "Fkbp5"  "Dnajb1"
## 
## $`response to phorbol 13-acetate 12-myristate`
## [1] "Fos"    "Btg2"   "Adam15"
## 
## $`cellular response to phorbol 13-acetate 12-myristate`
## [1] "Fos"    "Btg2"   "Adam15"
## 
## $`osteoclast differentiation`
## [1] "Fos"    "Junb"   "Bmp2"   "Klf10"  "Anxa2"  "Mafb"   "Pik3r1"
## 
## $`response to topologically incorrect protein`
## [1] "Hspb1"  "Hspa1a" "Manf"   "Hspa5"  "Clu"    "Serp2"  "Pik3r1"
## 
## $`SMAD protein signal transduction`
## [1] "Fos"    "Bmp2"   "Hfe"    "Bmper"  "Jun"    "Cited1"
## 
## $`negative regulation of protein serine/threonine kinase activity`
## [1] "Dusp1"    "Bmp2"     "Rgs2"     "Cdk5rap1" "Spry1"    "Gstp1"   
## 
## $`kidney development`
##  [1] "Cdkn1c" "Bmp2"   "Spry1"  "Hspa8"  "Bmper"  "Arg2"   "Cited1" "Robo2" 
##  [9] "Id3"    "Hoxb7"  "Irx1"  
## 
## $`prostaglandin transport`
## [1] "Ptgs2"   "Ptges"   "Pla2g4a" "Slco4a1"
## 
## $`inclusion body assembly`
## [1] "Hspa1b" "Hap1"   "Clu"    "Dnajb1"
## 
## $`regulation of hemopoiesis`
##  [1] "Fos"     "Hspb1"   "Bcl6"    "Cd83"    "Hspa1b"  "Klf10"   "Zfp36"  
##  [8] "Jun"     "Irf1"    "Mafb"    "Pik3r1"  "Tsc22d1"
## 
## $`regulation of transforming growth factor beta receptor signaling pathway`
## [1] "Cav2"    "Cdkn1c"  "Bmp2"    "Bambi"   "Spry1"   "Hspa5"   "Tsc22d1"
## 
## $`renal system development`
##  [1] "Cdkn1c" "Bmp2"   "Spry1"  "Hspa8"  "Bmper"  "Arg2"   "Cited1" "Robo2" 
##  [9] "Id3"    "Hoxb7"  "Irx1"  
## 
## $`negative regulation of cell-substrate adhesion`
## [1] "Ptprz1" "Bcl6"   "Acer2"  "Pik3r1" "Adam15"
## 
## $`cellular response to metal ion`
## [1] "Ptgs2"   "Fos"     "Junb"    "Chp2"    "Hfe"     "Hspa8"   "Jun"    
## [8] "Slc41a1"
## 
## $`regulation of cellular response to transforming growth factor beta stimulus`
## [1] "Cav2"    "Cdkn1c"  "Bmp2"    "Bambi"   "Spry1"   "Hspa5"   "Tsc22d1"
## 
## $`prostaglandin metabolic process`
## [1] "Ptgs2"   "Ptges"   "Pla2g4a" "Gstp1"   "Gstm1"  
## 
## $`prostanoid metabolic process`
## [1] "Ptgs2"   "Ptges"   "Pla2g4a" "Gstp1"   "Gstm1"  
## 
## $`regulation of transmembrane receptor protein serine/threonine kinase signaling pathway`
## [1] "Cav2"    "Cdkn1c"  "Bmp2"    "Bambi"   "Spry1"   "Hfe"     "Bmper"  
## [8] "Hspa5"   "Tsc22d1"
## 
## $`regulation of iron ion transport`
## [1] "Lcn2" "Hfe"  "Nos1"
## 
## $`negative regulation of cell-matrix adhesion`
## [1] "Bcl6"   "Acer2"  "Pik3r1" "Adam15"
## 
## $`negative regulation of cell-cell adhesion`
## [1] "Hspb1"  "Bcl6"   "Bmp2"   "Hfe"    "Arg2"   "Gstp1"  "Irf1"   "Pik3r1"
## 
## $`negative regulation of type 2 immune response`
## [1] "Bcl6" "Arg2" "Irf1"
## 
## $`response to steroid hormone`
##  [1] "Ptgs2"  "Fos"    "Junb"   "Hspa8"  "Zfp36"  "Jun"    "Cldn4"  "Gstp1" 
##  [9] "Pik3r1" "Por"   
## 
## $`response to unfolded protein`
## [1] "Hspb1"  "Hspa1a" "Manf"   "Hspa5"  "Serp2"  "Pik3r1"
## 
## $`regulation of membrane permeability`
## [1] "F11r"    "Hspa8"   "Ier3"    "Dynlt1f" "Cldn3"  
## 
## $`regulation of metal ion transport`
##  [1] "Ptgs2"    "Mcub"     "Lcn2"     "Hfe"      "Hap1"     "Trpc1"   
##  [7] "Cacna2d1" "Cx3cl1"   "Smim6"    "Nos1"     "Kcnc2"   
## 
## $`regulation of vesicle fusion`
## [1] "Syt5"    "Pla2g4a" "Rab3a"   "Anxa2"  
## 
## $`positive regulation of neuron projection development`
## [1] "Ptprz1"  "Hspb1"   "Rgs2"    "Scarf1"  "Hap1"    "Dynlt1f" "Hspa5"  
## [8] "Cx3cl1" 
## 
## $`carboxylic acid transport`
##  [1] "Ptgs2"    "Ptges"    "Rgs2"     "Slc6a7"   "Pla2g4a"  "Arg2"    
##  [7] "Slco4a1"  "Slc16a11" "Lrrc8c"   "Kcnk1"   
## 
## $`organic acid transport`
##  [1] "Ptgs2"    "Ptges"    "Rgs2"     "Slc6a7"   "Pla2g4a"  "Arg2"    
##  [7] "Slco4a1"  "Slc16a11" "Lrrc8c"   "Kcnk1"   
## 
## $`negative regulation of hemopoiesis`
## [1] "Hspb1"   "Bcl6"    "Irf1"    "Mafb"    "Pik3r1"  "Tsc22d1"
## 
## $`response to alcohol`
##  [1] "Fos"    "Btg2"   "Rgs2"   "Hspa8"  "Gstp1"  "Pmvk"   "Cldn3"  "Fkbp5" 
##  [9] "Adam15" "Kcnc2" 
## 
## $`membrane fusion`
## [1] "Syt5"    "Cav2"    "Mymx"    "Pla2g4a" "Rab3a"   "Anxa2"   "Stxbp6" 
## 
## $`amide metabolic process`
##  [1] "Degs2" "Aldob" "Hap1"  "Arg2"  "Gstp1" "Acer2" "Clu"   "Pmvk"  "Gstm1"
## [10] "Cmas"  "Gsta4"
## 
## $`vesicle fusion`
## [1] "Syt5"    "Cav2"    "Pla2g4a" "Rab3a"   "Anxa2"   "Stxbp6" 
## 
## $`negative regulation of apoptotic signaling pathway`
## [1] "Ptgs2"  "Hspb1"  "Hspa1b" "Ier3"   "Gstp1"  "Clu"    "Cx3cl1" "Pam16" 
## 
## $`organelle membrane fusion`
## [1] "Syt5"    "Cav2"    "Pla2g4a" "Rab3a"   "Anxa2"   "Stxbp6" 
## 
## $`nitric oxide biosynthetic process`
## [1] "Ptgs2"  "Dynll1" "Clu"    "Nos1"   "Por"   
## 
## $`prostaglandin secretion`
## [1] "Ptgs2"   "Ptges"   "Pla2g4a"
## 
## $`regulation of receptor binding`
## [1] "Hfe"    "Anxa2"  "Adam15"
## 
## $`negative regulation of alpha-beta T cell activation`
## [1] "Bcl6" "Hfe"  "Arg2" "Irf1"
## 
## $`regulation of neuron apoptotic process`
## [1] "Ptprz1" "Btg2"   "Lcn2"   "Jun"    "Cited1" "Tox3"   "Clu"    "Cx3cl1"
## [9] "Nos1"  
## 
## $`regulation of tumor necrosis factor production`
## [1] "Hspb1"  "Zfp36"  "Arg2"   "Gstp1"  "Clu"    "Cx3cl1" "Pik3r1"
## 
## $`regulation of tumor necrosis factor superfamily cytokine production`
## [1] "Hspb1"  "Zfp36"  "Arg2"   "Gstp1"  "Clu"    "Cx3cl1" "Pik3r1"
## 
## $`tumor necrosis factor production`
## [1] "Hspb1"  "Zfp36"  "Arg2"   "Gstp1"  "Clu"    "Cx3cl1" "Pik3r1"
## 
## $`regulation of monoatomic cation transmembrane transport`
## [1] "Mcub"     "Lcn2"     "Hap1"     "Trpc1"    "Cacna2d1" "Cx3cl1"   "Smim6"   
## [8] "Nos1"     "Kcnc2"   
## 
## $`regulation of protein kinase activity`
##  [1] "Hspb1"    "Dusp1"    "Cdkn1c"   "Sfn"      "Bmp2"     "Rgs2"    
##  [7] "Cdk5rap1" "Spry1"    "Sh3bp5"   "Gstp1"    "Map2k3"  
## 
## $`response to calcium ion`
## [1] "Fos"     "Junb"    "Chp2"    "Jun"     "Trpc1"   "Pla2g4a"
## 
## $`tumor necrosis factor superfamily cytokine production`
## [1] "Hspb1"  "Zfp36"  "Arg2"   "Gstp1"  "Clu"    "Cx3cl1" "Pik3r1"
## 
## $`nitric oxide metabolic process`
## [1] "Ptgs2"  "Dynll1" "Clu"    "Nos1"   "Por"   
## 
## $`cellular response to chemical stress`
## [1] "Ptgs2"  "Fos"    "Hspb1"  "Rhob"   "Lcn2"   "Hspa8"  "Jun"    "Fut8"  
## [9] "Lrrc8c"
## 
## $`cellular response to heat`
## [1] "Ptgs2"  "Hspa1b" "Hspa8"  "Dnajb1"
## 
## $`negative regulation of transforming growth factor beta receptor signaling pathway`
## [1] "Cav2"  "Bmp2"  "Bambi" "Spry1" "Hspa5"
## 
## $`reactive nitrogen species metabolic process`
## [1] "Ptgs2"  "Dynll1" "Clu"    "Nos1"   "Por"   
## 
## $`negative regulation of response to external stimulus`
##  [1] "Dusp1"  "Sfn"    "Zfp36"  "Ier3"   "Arg2"   "Gstp1"  "Anxa2"  "Robo2" 
##  [9] "Cx3cl1" "Cldn3" 
## 
## $`regulation of acute inflammatory response`
## [1] "Ptgs2"  "Ptges"  "C2cd4b" "Gstp1" 
## 
## $`metanephros development`
## [1] "Spry1"  "Cited1" "Robo2"  "Id3"    "Irx1"  
## 
## $`regulation of animal organ morphogenesis`
## [1] "Bmp2"   "Wnt10a" "Spry1"  "Robo2"  "Hoxb7" 
## 
## $`negative regulation of transmembrane receptor protein serine/threonine kinase signaling pathway`
## [1] "Cav2"  "Bmp2"  "Bambi" "Spry1" "Bmper" "Hspa5"
## 
## $`sulfur compound biosynthetic process`
## [1] "B3gnt7" "Hs3st6" "Gstp1"  "Ndst4"  "Gstm1"  "Mri1"  
## 
## $`organ induction`
## [1] "Bmp2"  "Spry1" "Robo2"
## 
## $`regulation of calcium ion transport`
## [1] "Ptgs2"    "Mcub"     "Hap1"     "Trpc1"    "Cacna2d1" "Cx3cl1"   "Smim6"   
## [8] "Nos1"    
## 
## $`negative regulation of leukocyte cell-cell adhesion`
## [1] "Hspb1" "Bcl6"  "Hfe"   "Arg2"  "Gstp1" "Irf1" 
## 
## $`p38MAPK cascade`
## [1] "Dusp1"  "Bmp2"   "Zfp36"  "Map2k3"
## 
## $`myeloid leukocyte differentiation`
## [1] "Fos"    "Junb"   "Bmp2"   "Klf10"  "Jun"    "Anxa2"  "Mafb"   "Pik3r1"
## 
## $`muscle cell proliferation`
## [1] "Ptgs2"  "Fos"    "Cav2"   "Bmp2"   "Jun"    "Gstp1"  "Cx3cl1" "Pik3r1"
## 
## $`response to glucocorticoid`
## [1] "Ptgs2"  "Fos"    "Zfp36"  "Gstp1"  "Pik3r1" "Por"   
## 
## $`positive regulation of cation transmembrane transport`
## [1] "Lcn2"   "Hap1"   "Trpc1"  "Cx3cl1" "Nos1"   "Kcnc2" 
## 
## $`positive regulation of transmembrane receptor protein serine/threonine kinase signaling pathway`
## [1] "Cdkn1c"  "Bmp2"    "Hfe"     "Bmper"   "Tsc22d1"
## 
## $`regulation of ATP-dependent activity`
## [1] "Aldob"  "Mcm2"   "Smim6"  "Dnajb1"
## 
## $`fatty acid biosynthetic process`
## [1] "Ptgs2"   "Ptges"   "Lipg"    "Pla2g4a" "Gstp1"   "Gstm1"  
## 
## $`regulation of blood pressure`
## [1] "Ptgs2"  "F11r"   "Ier3"   "Manf"   "Pik3r1" "Nos1"   "Klk1b3"
## 
## $`regulation of leukocyte differentiation`
## [1] "Fos"    "Hspb1"  "Bcl6"   "Cd83"   "Klf10"  "Jun"    "Irf1"   "Mafb"  
## [9] "Pik3r1"
## 
## $`glycoprotein metabolic process`
## [1] "Bmp2"    "B3gnt7"  "Tmtc1"   "Hs3st6"  "Acer2"   "Galnt3"  "Ndst4"  
## [8] "St3gal4" "Fut8"   
## 
## $`response to tumor necrosis factor`
## [1] "Ptgs2"  "Fos"    "Hspa1b" "Traf1"  "Zfp36"  "Irf1"   "Cx3cl1"
## 
## $`cell-cell adhesion via plasma-membrane adhesion molecules`
## [1] "Bmp2"   "Scarf1" "Cldn4"  "Pcdh1"  "Robo2"  "Cx3cl1" "Cldn3" 
## 
## $`regulation of monoatomic ion transmembrane transport`
## [1] "Mcub"     "Lcn2"     "Hap1"     "Trpc1"    "Cacna2d1" "Cx3cl1"   "Smim6"   
## [8] "Nos1"     "Kcnc2"   
## 
## $`response to ethanol`
## [1] "Fos"   "Rgs2"  "Hspa8" "Gstp1" "Cldn3" "Kcnc2"
## 
## $`female pregnancy`
## [1] "Ptgs2"   "Fos"     "Junb"    "Rgs2"    "Hfe"     "Cldn4"   "Pla2g4a"
## 
## $`iron ion transport`
## [1] "Lcn2" "Hfe"  "Fth1" "Nos1"
## 
## $`cell-substrate adhesion`
## [1] "Ptprz1" "Frem1"  "Bcl6"   "Limch1" "Cspg5"  "Acer2"  "Anxa2"  "Pik3r1"
## [9] "Adam15"
## 
## $`positive regulation of vesicle fusion`
## [1] "Syt5"    "Pla2g4a" "Anxa2"  
## 
## $`negative regulation of muscle contraction`
## [1] "Ptgs2" "Rgs2"  "Arg2" 
## 
## $`response to toxic substance`
## [1] "Ptgs2" "Fos"   "Ptges" "Lcn2"  "Pon2"  "Gstp1" "Kcnc2"
## 
## $`regulation of epidermis development`
## [1] "Sfn"   "Etv4"  "Zfp36" "Nab2" 
## 
## $`positive regulation of endothelial cell migration`
## [1] "Ptgs2"  "Hspb1"  "Rhob"   "Lcn2"   "Map2k3"
## 
## $`regulation of cell-substrate adhesion`
## [1] "Ptprz1" "Bcl6"   "Limch1" "Cspg5"  "Acer2"  "Pik3r1" "Adam15"
## 
## $`regulation of myeloid cell differentiation`
## [1] "Fos"    "Hspa1b" "Klf10"  "Zfp36"  "Jun"    "Mafb"   "Pik3r1"
## 
## $`macrophage activation`
## [1] "Mcub"    "Jun"     "Pla2g4a" "Clu"     "Cx3cl1" 
## 
## $`cell-matrix adhesion`
## [1] "Frem1"  "Bcl6"   "Limch1" "Acer2"  "Anxa2"  "Pik3r1" "Adam15"
## 
## $`maternal process involved in female pregnancy`
## [1] "Ptgs2"   "Junb"    "Rgs2"    "Pla2g4a"
## 
## $`osteoblast differentiation`
## [1] "Ache"   "Junb"   "Bmp2"   "Bambi"  "Tent5a" "Cited1" "Id3"   
## 
## $`glycoprotein biosynthetic process`
## [1] "B3gnt7"  "Tmtc1"   "Hs3st6"  "Acer2"   "Galnt3"  "Ndst4"   "St3gal4"
## [8] "Fut8"   
## 
## $`plasma membrane organization`
## [1] "Cav2"  "Mymx"  "Rab3a" "Anxa2" "Clu"   "Mafb" 
## 
## $`response to starvation`
## [1] "Fos"   "Hfe"   "Klf10" "Hspa8" "Zfp36" "Jun"   "Hspa5"
## 
## $`prostaglandin biosynthetic process`
## [1] "Ptgs2"   "Ptges"   "Pla2g4a"
## 
## $`regulation of animal organ formation`
## [1] "Bmp2"  "Spry1" "Robo2"
## 
## $`prostanoid biosynthetic process`
## [1] "Ptgs2"   "Ptges"   "Pla2g4a"
## 
## $`cellular response to cadmium ion`
## [1] "Fos"   "Hspa8" "Jun"  
## 
## $`icosanoid transport`
## [1] "Ptgs2"   "Ptges"   "Pla2g4a" "Slco4a1"
## 
## $`organic anion transport`
##  [1] "Ptgs2"    "Ptges"    "Rgs2"     "Slc6a7"   "Pla2g4a"  "Arg2"    
##  [7] "Slco4a1"  "Slc16a11" "Lrrc8c"   "Kcnk1"   
## 
## $`regulation of endothelial cell migration`
## [1] "Ptgs2"  "Hspb1"  "Rhob"   "Lcn2"   "Bmper"  "Map2k3"
## 
## $`positive regulation of monoatomic ion transmembrane transport`
## [1] "Lcn2"   "Hap1"   "Trpc1"  "Cx3cl1" "Nos1"   "Kcnc2" 
## 
## $`acute inflammatory response`
## [1] "Ptgs2"  "Ptges"  "Hfe"    "C2cd4b" "Gstp1" 
## 
## $`positive regulation of fat cell differentiation`
## [1] "Ptgs2"  "Bmp2"   "Zfp36"  "Vstm2a"
## 
## $`neuron apoptotic process`
## [1] "Ptprz1" "Btg2"   "Lcn2"   "Jun"    "Cited1" "Tox3"   "Clu"    "Cx3cl1"
## [9] "Nos1"  
## 
## $decidualization
## [1] "Ptgs2"   "Junb"    "Pla2g4a"
## 
## $`cardiac epithelial to mesenchymal transition`
## [1] "Bmp2"   "Spry1"  "Adam15"
## 
## $`response to corticosteroid`
## [1] "Ptgs2"  "Fos"    "Zfp36"  "Gstp1"  "Pik3r1" "Por"   
## 
## $`regulation of neurogenesis`
##  [1] "Ptprz1"  "Ache"    "Btg2"    "Bmp2"    "Hap1"    "Anxa2"   "Robo2"  
##  [8] "Cx3cl1"  "Rassf10" "Nos1"   
## 
## $`negative regulation of leukocyte differentiation`
## [1] "Hspb1"  "Bcl6"   "Irf1"   "Mafb"   "Pik3r1"
## 
## $`regulation of type 2 immune response`
## [1] "Bcl6" "Arg2" "Irf1"
## 
## $`regulation of supramolecular fiber organization`
## [1] "Hspa1a" "Hspa1b" "F11r"   "Limch1" "Hspa8"  "Clu"    "Capg"   "Cx3cl1"
## [9] "Pik3r1"
## 
## $`regulation of alpha-beta T cell activation`
## [1] "Bcl6" "Cd83" "Hfe"  "Arg2" "Irf1"
## 
## $`regulation of fibroblast proliferation`
## [1] "Ptprz1" "Jun"    "Gstp1"  "Anxa2"  "Fth1"  
## 
## $`positive regulation of lipid localization`
## [1] "Ptges"   "Lipg"    "Vstm2a"  "Pla2g4a" "Anxa2"  
## 
## $`organelle fusion`
## [1] "Syt5"    "Cav2"    "Pla2g4a" "Rab3a"   "Anxa2"   "Stxbp6" 
## 
## $`negative regulation of calcium ion transport`
## [1] "Ptgs2" "Mcub"  "Smim6" "Nos1" 
## 
## $`regulation of cellular response to growth factor stimulus`
## [1] "Cav2"    "Cdkn1c"  "Bmp2"    "Bambi"   "Spry1"   "Bmper"   "Hspa5"  
## [8] "Tsc22d1"
## 
## $`response to xenobiotic stimulus`
##  [1] "Ptgs2" "Fos"   "Lcn2"  "Hspa8" "Jun"   "Gstp1" "Acer2" "Gstm1" "Gsta4"
## [10] "Nos1" 
## 
## $`cellular response to oxidative stress`
## [1] "Fos"   "Hspb1" "Rhob"  "Lcn2"  "Hspa8" "Jun"   "Fut8" 
## 
## $`multi-organism reproductive process`
## [1] "Ptgs2"   "Fos"     "Junb"    "Rgs2"    "Hfe"     "Cldn4"   "Pla2g4a"
## 
## $`positive regulation of protein binding`
## [1] "Bmp2"  "Bambi" "Hfe"   "Anxa2"
## 
## $`positive regulation of nucleocytoplasmic transport`
## [1] "Ptgs2"  "Chp2"   "Sfn"    "Pik3r1"
## 
## $`regulation of calcium ion transmembrane transport`
## [1] "Mcub"     "Hap1"     "Trpc1"    "Cacna2d1" "Cx3cl1"   "Smim6"   
## 
## $`regulation of cell-matrix adhesion`
## [1] "Bcl6"   "Limch1" "Acer2"  "Pik3r1" "Adam15"
## 
## $`positive regulation of proteolysis`
## [1] "Hspa1a" "Hspa1b" "Hspa8"  "Cldn4"  "Acer2"  "Anxa2"  "Clu"    "Cldn3" 
## 
## $`regulation of lens fiber cell differentiation`
## [1] "Cdkn1c" "Spry1" 
## 
## $`regulation of lipid localization`
## [1] "Ptges"   "Lipg"    "Vstm2a"  "Pla2g4a" "Anxa2"   "Ttc39b" 
## 
## $`multi-multicellular organism process`
## [1] "Ptgs2"   "Fos"     "Junb"    "Rgs2"    "Hfe"     "Cldn4"   "Pla2g4a"
## 
## $`regulation of protein serine/threonine kinase activity`
## [1] "Dusp1"    "Sfn"      "Bmp2"     "Rgs2"     "Cdk5rap1" "Spry1"    "Gstp1"   
## 
## $`positive regulation of response to wounding`
## [1] "Scarf1"  "Cldn4"   "Cldn3"   "St3gal4"
## 
## $`response to mechanical stimulus`
## [1] "Ptgs2" "Fos"   "Btg2"  "F11r"  "Jun"   "Irf1"  "Nos1" 
## 
## $`skeletal system morphogenesis`
## [1] "Pappa2" "Frem1"  "Hoxc4"  "Hoxb2"  "Hoxb7"  "Por"    "Nab2"  
## 
## $`hormone metabolic process`
## [1] "Iyd"     "Bmp2"    "Hfe"     "Slco4a1" "Cpq"     "Akr1b8"  "Por"    
## 
## $`cellular modified amino acid metabolic process`
## [1] "Iyd"     "Gstp1"   "Slco4a1" "Cpq"     "Gstm1"   "Gsta4"  
## 
## $`regulation of smooth muscle cell proliferation`
## [1] "Ptgs2"  "Bmp2"   "Jun"    "Gstp1"  "Cx3cl1" "Pik3r1"
## 
## $`phenol-containing compound metabolic process`
## [1] "Iyd"     "Trpc1"   "Slco4a1" "Cited1"  "Cpq"    
## 
## $`regulation of nucleocytoplasmic transport`
## [1] "Ptgs2"  "Chp2"   "Sfn"    "Ier3"   "Pik3r1"
## 
## $`fat cell differentiation`
## [1] "Ptgs2"  "Bmp2"   "Rgs2"   "Zfp36"  "Vstm2a" "Psmb8"  "Lrrc8c"
## 
## $`positive regulation of phosphatase activity`
## [1] "Chp2" "Bmp2"
## 
## $`regulation of microtubule nucleation`
## [1] "Hspa1a" "Hspa1b"
## 
## $`regulation of fever generation`
## [1] "Ptgs2" "Ptges"
## 
## $`nitric oxide-cGMP-mediated signaling`
## [1] "Nos1"  "Kcnc2"
## 
## $`negative regulation of erythrocyte differentiation`
## [1] "Zfp36" "Mafb" 
## 
## $`negative regulation of inclusion body assembly`
## [1] "Hspa1b" "Dnajb1"
## 
## $`positive regulation of unsaturated fatty acid biosynthetic process`
## [1] "Ptgs2"   "Pla2g4a"
## 
## $`regulation of response to endoplasmic reticulum stress`
## [1] "Manf"   "Hspa5"  "Clu"    "Pik3r1"
## 
## $`thyroid hormone metabolic process`
## [1] "Iyd"     "Slco4a1" "Cpq"    
## 
## $`response to interleukin-4`
## [1] "Mcm2"   "Hspa5"  "Cited1"
## 
## $`negative regulation of CD4-positive, alpha-beta T cell activation`
## [1] "Bcl6" "Arg2" "Irf1"
## 
## $`negative regulation of leukocyte activation`
## [1] "Hspb1"  "Bcl6"   "Hfe"    "Arg2"   "Irf1"   "Cx3cl1"
## 
## $`sulfur compound metabolic process`
## [1] "B3gnt7" "Hs3st6" "Gstp1"  "Pmvk"   "Ndst4"  "Gstm1"  "Gsta4"  "Mri1"  
## 
## $`response to oxidative stress`
## [1] "Ptgs2" "Fos"   "Hspb1" "Rhob"  "Lcn2"  "Hspa8" "Jun"   "Gstp1" "Fut8" 
## 
## $`leukocyte cell-cell adhesion`
## [1] "Hspb1"   "Bcl6"    "Cd83"    "F11r"    "Hfe"     "Arg2"    "Gstp1"  
## [8] "Irf1"    "St3gal4"
## 
## $`cellular response to calcium ion`
## [1] "Fos"  "Junb" "Chp2" "Jun" 
## 
## $`specification of animal organ identity`
## [1] "Bmp2"  "Spry1" "Robo2"
## 
## $`developmental induction`
## [1] "Bmp2"  "Spry1" "Robo2"
## 
## $`positive regulation of SMAD protein signal transduction`
## [1] "Bmp2"  "Hfe"   "Bmper"
## 
## $`positive regulation of animal organ morphogenesis`
## [1] "Bmp2"  "Spry1" "Robo2"
## 
## $`response to endoplasmic reticulum stress`
## [1] "Manf"   "Jun"    "Hspa5"  "Clu"    "Nrbf2"  "Serp2"  "Pik3r1"
## 
## $`cellular response to tumor necrosis factor`
## [1] "Fos"    "Hspa1b" "Traf1"  "Zfp36"  "Irf1"   "Cx3cl1"
## 
## $`negative regulation of T cell activation`
## [1] "Hspb1" "Bcl6"  "Hfe"   "Arg2"  "Irf1" 
## 
## $`negative regulation of locomotion`
## [1] "Dusp1"  "Rhob"   "Limch1" "Gstp1"  "Robo2"  "Cx3cl1" "Cldn3"  "Adam15"
## 
## $`smooth muscle cell proliferation`
## [1] "Ptgs2"  "Bmp2"   "Jun"    "Gstp1"  "Cx3cl1" "Pik3r1"
## 
## $`regulation of prostaglandin biosynthetic process`
## [1] "Ptgs2"   "Pla2g4a"
## 
## $`macrophage proliferation`
## [1] "Clu"    "Cx3cl1"
## 
## $`chaperone-mediated autophagy`
## [1] "Hspa8" "Clu"  
## 
## $`regulation of nucleotide-binding domain, leucine rich repeat containing receptor signaling pathway`
## [1] "Hspa1b"  "Slc15a2"
## 
## $`negative regulation of receptor binding`
## [1] "Hfe"    "Adam15"
## 
## $`positive regulation of non-motile cilium assembly`
## [1] "Hap1"   "Dynll1"
## 
## $`regulation of CD4-positive, alpha-beta T cell activation`
## [1] "Bcl6" "Cd83" "Arg2" "Irf1"
## 
## $`regulation of regulatory T cell differentiation`
## [1] "Hspb1" "Bcl6"  "Irf1" 
## 
## $`regulation of apoptotic signaling pathway`
## [1] "Ptgs2"  "Hspb1"  "Hspa1b" "Traf1"  "Ier3"   "Gstp1"  "Clu"    "Cx3cl1"
## [9] "Pam16" 
## 
## $`import across plasma membrane`
## [1] "Rgs2"    "Lcn2"    "Slc6a7"  "Arg2"    "Lrrc8c"  "Slc15a2"
## 
## $`nephron tubule morphogenesis`
## [1] "Bmp2"   "Cited1" "Hoxb7"  "Irx1"  
## 
## $`nucleocytoplasmic transport`
## [1] "Ptgs2"   "Chp2"    "Sfn"     "Hspa8"   "Ier3"    "Cited1"  "Pik3r1" 
## [8] "Htatip2"
## 
## $`nuclear transport`
## [1] "Ptgs2"   "Chp2"    "Sfn"     "Hspa8"   "Ier3"    "Cited1"  "Pik3r1" 
## [8] "Htatip2"
## 
## $`response to progesterone`
## [1] "Fos"   "Hspa8" "Cldn4"
## 
## $`neuroinflammatory response`
## [1] "Ptgs2"  "Jun"    "Clu"    "Cx3cl1"
## 
## $regionalization
## [1] "Btg2"  "Bmp2"  "Spry1" "Robo2" "Mafb"  "Hoxc4" "Hoxb2" "Hoxb7" "Irx1" 
## 
## $`tissue regeneration`
## [1] "Mcub"   "Mymx"   "Cpq"    "Adam15"
## 
## $`nephron epithelium morphogenesis`
## [1] "Bmp2"   "Cited1" "Hoxb7"  "Irx1"  
## 
## $`positive regulation of dephosphorylation`
## [1] "Chp2" "Bmp2"
## 
## $`positive regulation of nuclear-transcribed mRNA poly(A) tail shortening`
## [1] "Btg2"  "Zfp36"
## 
## $`regulation of metallopeptidase activity`
## [1] "Cldn4" "Cldn3"
## 
## $`maternal placenta development`
## [1] "Ptgs2"   "Junb"    "Pla2g4a"
## 
## $`negative regulation of chemotaxis`
## [1] "Dusp1" "Gstp1" "Robo2"
## 
## $`positive regulation of release of sequestered calcium ion into cytosol`
## [1] "Hap1"   "Trpc1"  "Cx3cl1"
## 
## $`alcohol metabolic process`
## [1] "Bmp2"    "Degs2"   "Pla2g4a" "Acer2"   "Ttc39b"  "Pmvk"    "Akr1b8" 
## [8] "Por"    
## 
## $`regulation of osteoclast differentiation`
## [1] "Fos"    "Klf10"  "Mafb"   "Pik3r1"
## 
## $`axo-dendritic transport`
## [1] "Hspb1" "Hspa8" "Hap1"  "Rab17"
## 
## $`nephron morphogenesis`
## [1] "Bmp2"   "Cited1" "Hoxb7"  "Irx1"  
## 
## $`positive regulation of leukocyte differentiation`
## [1] "Fos"   "Bcl6"  "Cd83"  "Klf10" "Jun"   "Irf1" 
## 
## $`positive regulation of hemopoiesis`
## [1] "Fos"   "Bcl6"  "Cd83"  "Klf10" "Jun"   "Irf1" 
## 
## $`regulation of myeloid leukocyte differentiation`
## [1] "Fos"    "Klf10"  "Jun"    "Mafb"   "Pik3r1"
## 
## $`fibroblast proliferation`
## [1] "Ptprz1" "Jun"    "Gstp1"  "Anxa2"  "Fth1"  
## 
## $`response to reactive oxygen species`
## [1] "Fos"   "Rhob"  "Lcn2"  "Hspa8" "Jun"   "Gstp1"
## 
## $`protein glycosylation`
## [1] "B3gnt7"  "Tmtc1"   "Acer2"   "Galnt3"  "St3gal4" "Fut8"   
## 
## $`macromolecule glycosylation`
## [1] "B3gnt7"  "Tmtc1"   "Acer2"   "Galnt3"  "St3gal4" "Fut8"   
## 
## $`carboxylic acid transmembrane transport`
## [1] "Rgs2"     "Slc6a7"   "Arg2"     "Slc16a11" "Lrrc8c"  
## 
## $`cellular response to epidermal growth factor stimulus`
## [1] "Fos"   "Zfp36" "Gstp1"
## 
## $`negative regulation of cell activation`
## [1] "Hspb1"  "Bcl6"   "Hfe"    "Arg2"   "Irf1"   "Cx3cl1"
## 
## $`fever generation`
## [1] "Ptgs2" "Ptges"
## 
## $`membrane raft assembly`
## [1] "Cav2"  "Anxa2"
## 
## $`regulation of T-helper 2 cell differentiation`
## [1] "Bcl6" "Irf1"
## 
## $`hepoxilin metabolic process`
## [1] "Gstp1" "Gstm1"
## 
## $`hepoxilin biosynthetic process`
## [1] "Gstp1" "Gstm1"
## 
## $`response to parathyroid hormone`
## [1] "Fos"    "Cited1"
## 
## $`positive regulation of glial cell migration`
## [1] "Ptprz1" "Cx3cl1"
## 
## $`regulation of unsaturated fatty acid biosynthetic process`
## [1] "Ptgs2"   "Pla2g4a"
## 
## $`renal tubule morphogenesis`
## [1] "Bmp2"   "Cited1" "Hoxb7"  "Irx1"  
## 
## $`icosanoid metabolic process`
## [1] "Ptgs2"   "Ptges"   "Pla2g4a" "Gstp1"   "Gstm1"  
## 
## $`positive regulation of intracellular transport`
## [1] "Ptgs2"  "Chp2"   "Sfn"    "Anxa2"  "Pik3r1"
## 
## $`regulation of intracellular transport`
## [1] "Ptgs2"  "Chp2"   "Sfn"    "Hap1"   "Ier3"   "Anxa2"  "Pik3r1"
## 
## $`regulation of lipid transport`
## [1] "Ptges"   "Lipg"    "Pla2g4a" "Anxa2"   "Ttc39b" 
## 
## $`monocarboxylic acid biosynthetic process`
## [1] "Ptgs2"   "Ptges"   "Lipg"    "Pla2g4a" "Gstp1"   "Gstm1"  
## 
## $`regulatory T cell differentiation`
## [1] "Hspb1" "Bcl6"  "Irf1" 
## 
## $`unsaturated fatty acid metabolic process`
## [1] "Ptgs2"   "Ptges"   "Pla2g4a" "Gstp1"   "Gstm1"  
## 
## $`organic acid transmembrane transport`
## [1] "Rgs2"     "Slc6a7"   "Arg2"     "Slc16a11" "Lrrc8c"  
## 
## $`calcium ion transport`
## [1] "Ptgs2"    "Mcub"     "Tmem37"   "Hap1"     "Trpc1"    "Cacna2d1" "Cx3cl1"  
## [8] "Smim6"    "Nos1"    
## 
## $`cellular response to reactive oxygen species`
## [1] "Fos"   "Rhob"  "Lcn2"  "Hspa8" "Jun"  
## 
## $`positive regulation of prostaglandin secretion`
## [1] "Ptges"   "Pla2g4a"
## 
## $`regulation of odontogenesis of dentin-containing tooth`
## [1] "Bmp2"   "Wnt10a"
## 
## $`regulation of nuclear-transcribed mRNA poly(A) tail shortening`
## [1] "Btg2"  "Zfp36"

3.2 Downregulation Pathway

# Arrange and filter DEGs

df <- type_markers %>% arrange(desc(avg_log2FC))

df2 <- df %>% filter(p_val_adj < 0.05)

DEG_list <- df2

markers1down <- DEG_list %>% rownames_to_column(var="SYMBOL")

ENTREZ_list <- bitr(
  geneID = rownames(DEG_list),
  fromType = "SYMBOL",
  toType = "ENTREZID",
  OrgDb = org.Mm.eg.db
)
## 'select()' returned 1:1 mapping between keys and columns
## Warning in bitr(geneID = rownames(DEG_list), fromType = "SYMBOL", toType =
## "ENTREZID", : 3.19% of input gene IDs are fail to map...
markers1down <- ENTREZ_list %>% inner_join(markers1down, by = "SYMBOL")

markers1down <- markers1down %>% dplyr::filter(p_val_adj < 0.05)

pos.markers1down <- markers1down %>% dplyr::filter(avg_log2FC < 0) %>% arrange(desc(abs(avg_log2FC)))

pos.ranks1down <- pos.markers1down$ENTREZID[abs(pos.markers1down$avg_log2FC) > 0.4]

# GO enrichment for downregulated genes
pos_go1down <- enrichGO(
  gene = pos.ranks1down,
  OrgDb = org.Mm.eg.db,
  ont = "BP",
  readable = TRUE
)

pos_go1down
## #
## # over-representation test
## #
## #...@organism     Mus musculus 
## #...@ontology     BP 
## #...@keytype      ENTREZID 
## #...@gene     chr [1:65] "268709" "54156" "18703" "15395" "12709" "192201" "27528" ...
## #...pvalues adjusted by 'BH' with cutoff <0.05 
## #...22 enriched terms found
## 'data.frame':    22 obs. of  12 variables:
##  $ ID            : chr  "GO:0070374" "GO:0042060" "GO:0022612" "GO:0032148" ...
##  $ Description   : chr  "positive regulation of ERK1 and ERK2 cascade" "wound healing" "gland morphogenesis" "activation of protein kinase B activity" ...
##  $ GeneRatio     : chr  "6/61" "7/61" "5/61" "3/61" ...
##  $ BgRatio       : chr  "227/28928" "394/28928" "163/28928" "29/28928" ...
##  $ RichFactor    : num  0.0264 0.0178 0.0307 0.1034 0.0909 ...
##  $ FoldEnrichment: num  12.53 8.43 14.55 49.06 43.11 ...
##  $ zScore        : num  8.02 6.82 7.97 11.9 11.13 ...
##  $ pvalue        : num  8.46e-06 1.91e-05 2.46e-05 3.13e-05 4.65e-05 ...
##  $ p.adjust      : num  0.0106 0.0106 0.0106 0.0106 0.0106 ...
##  $ qvalue        : num  0.00817 0.00817 0.00817 0.00817 0.00817 ...
##  $ geneID        : chr  "Fgfbp3/Dusp15/Gas6/Egf/Fgf1/Notch2" "Plau/Plet1/Gas6/Cldn19/Fgf1/Notch2/Kng1" "Plau/Ntn1/Sostdc1/Fgf1/Notch2" "Gas6/Fgf1/Notch2" ...
##  $ Count         : int  6 7 5 3 3 3 3 6 6 3 ...
## #...Citation
## G Yu. Thirteen years of clusterProfiler. The Innovation. 2024, 5(6):100722
# Visualization
chart1down <- dotplot(pos_go1down) +
  ggtitle("type 1 downregulated control") +
  theme_classic() +
  theme(
    plot.title = element_text(hjust = 0.5),
    legend.position = "left",
    axis.text.y = element_text(hjust = 0, size = 10)
  ) +
  scale_y_discrete(
    position = "right",
    labels = function(x) str_wrap(x, width = 25)
  )
## Scale for y is already present.
## Adding another scale for y, which will replace the existing scale.
chart1down

3.2.1 Downreg Gene View

pathway_type1down <- strsplit(pos_go1down$geneID, "/")

names(pathway_type1down) <- pos_go1down$Description 

pathway_type1down
## $`positive regulation of ERK1 and ERK2 cascade`
## [1] "Fgfbp3" "Dusp15" "Gas6"   "Egf"    "Fgf1"   "Notch2"
## 
## $`wound healing`
## [1] "Plau"   "Plet1"  "Gas6"   "Cldn19" "Fgf1"   "Notch2" "Kng1"  
## 
## $`gland morphogenesis`
## [1] "Plau"    "Ntn1"    "Sostdc1" "Fgf1"    "Notch2" 
## 
## $`activation of protein kinase B activity`
## [1] "Gas6"   "Fgf1"   "Notch2"
## 
## $`hepatocyte proliferation`
## [1] "Plau"   "Fgf1"   "Notch2"
## 
## $`epithelial cell proliferation involved in liver morphogenesis`
## [1] "Plau"   "Fgf1"   "Notch2"
## 
## $`liver morphogenesis`
## [1] "Plau"   "Fgf1"   "Notch2"
## 
## $`regulation of ERK1 and ERK2 cascade`
## [1] "Fgfbp3" "Dusp15" "Gas6"   "Egf"    "Fgf1"   "Notch2"
## 
## $`regulation of cellular response to growth factor stimulus`
## [1] "Nrep"    "Fgf9"    "Fgfbp3"  "Sostdc1" "Fgf1"    "Notch2" 
## 
## $`renal absorption`
## [1] "Umod"   "Gas6"   "Cldn19"
## 
## $`negative regulation of proteolysis`
## [1] "Ide"   "Plau"  "Timp3" "Gas6"  "Kng1"  "Anks1"
## 
## $`ERK1 and ERK2 cascade`
## [1] "Fgfbp3" "Dusp15" "Gas6"   "Egf"    "Fgf1"   "Notch2"
## 
## $`regulation of epithelial cell proliferation`
## [1] "Plau"   "Fgf9"   "Egf"    "Fmc1"   "Fgf1"   "Notch2"
## 
## $`positive regulation of protein kinase activity`
## [1] "Vldlr"  "Gas6"   "Egf"    "Fgf1"   "Notch2"
## 
## $`chaperone-mediated protein folding`
## [1] "Fkbp11"   "Umod"     "Pdcd5-ps"
## 
## $`negative regulation of wound healing`
## [1] "Plau"   "Cldn19" "Kng1"  
## 
## $`positive regulation of kinase activity`
## [1] "Vldlr"  "Gas6"   "Egf"    "Fgf1"   "Notch2"
## 
## $`intracellular chloride ion homeostasis`
## [1] "Ckb"  "Umod"
## 
## $`vasculogenesis involved in coronary vascular morphogenesis`
## [1] "Fgf9" "Fgf1"
## 
## $`activation of protein kinase activity`
## [1] "Gas6"   "Fgf1"   "Notch2"
## 
## $`intracellular monoatomic anion homeostasis`
## [1] "Ckb"  "Umod"
## 
## $`fibroblast growth factor receptor signaling pathway`
## [1] "Fgf9"   "Fgfbp3" "Fgf1"
# Start filtering process here
# Maybe use genes you discover from Upset Plot
# Maybe  try with subtypes fitler genes


gyarmati <- c("Fabp3", "Ccn1", "Foxq1", "Cxcl12", "Vash2", "Pamr1", "Vegfd", "Ccn3", "Bmp3", "Fgf9", "Spp1", "Wnt10a", "Sfrp1", "Tcf4", "Pappa2", "Unc5d", "Sema3c", "Robo2", "Slit2", "Egfl6", "Hgfac", "Pdgfc", "Megf9", "Frem1", "Thsd4", "Spock2", "Mmp14", "Adamtsl2", "Car8", "Irx1", "Irx2", "Hoxd11", "Hoxc4", "Etv1")

df3 <- type_markers %>%
  filter(gene %in% gyarmati) %>%
  arrange(desc(avg_log2FC))
  
df3 <- rownames(df3)

df <- AverageExpression(
  object = SO4,
  features = df3,
  group.by = 'treatment'
)$RNA
## As of Seurat v5, we recommend using AggregateExpression to perform pseudo-bulk analysis.
## Names of identity class contain underscores ('_'), replacing with dashes ('-')
## This message is displayed once per session.
df
## 25 x 2 sparse Matrix of class "dgCMatrix"
##               control     low-salt
## Pappa2   6.591097e+35 1.304159e+66
## Frem1    6.673899e-01 3.198223e+00
## Wnt10a   2.273670e+00 7.980575e+00
## Robo2    6.943374e+15 1.199490e+20
## Hoxc4    8.302147e-01 1.078682e+00
## Irx1     5.138850e+16 3.271063e+20
## Megf9    1.427964e+02 6.102353e+03
## Irx2     2.409596e+10 5.795259e+09
## Etv1     2.207814e+01 6.491579e+01
## Bmp3     2.234102e+07 1.108405e+09
## Vegfd    3.172421e+00 5.166964e+00
## Sfrp1    8.596244e+87 3.679007e+88
## Vash2    3.939904e+00 3.543367e+01
## Spock2   8.860768e+01 8.436720e-01
## Hgfac    1.099643e+00 1.705886e+00
## Thsd4    3.184754e+02 1.560983e+02
## Adamtsl2 2.437454e+00 9.787557e+00
## Mmp14    2.911076e+00 4.862623e+00
## Hoxd11   2.703429e+00 4.963274e+00
## Pamr1    7.920054e+05 7.033070e+07
## Slit2    4.645370e+06 1.757724e+09
## Cxcl12   1.795344e+00 1.488615e+00
## Fgf9     7.375148e+04 4.247585e+06
## Fabp3    8.119897e+06 1.642683e+06
## Egfl6    1.330964e+01 3.049626e+00
df <- as.data.frame(df)

df <- t(scale(t(df)))

# convert df to tidy format

# only use about 10-15 genes

df_tidy <- df %>%
  as.data.frame() %>%
  rownames_to_column(var = "Gene") %>%
  pivot_longer(cols = -Gene, names_to = "treatment", values_to = "Expression")
               
# Graph with geom_tile

df_tidy$treatment <- factor(df_tidy$treatment, levels = c("control","low-salt"))

f4 <- ggplot(df_tidy, aes(x = treatment, y = Gene, fill = Expression)) +
  geom_tile(color = "black", size = 0.2, width = 0.97, height = 0.90) +  # Adds thin border and spacing between tiles
  scale_fill_distiller(palette = "RdYlBu") +
  theme_minimal() +
  theme(
    axis.text = element_text(size = 12, color = "black"),
    axis.title = element_text(size = 14),
    axis.ticks = element_blank(),         # Removes tick marks
    panel.grid = element_blank(),          # Removes grid lines
    
    legend.position = "bottom",            # Centers the legend below the plot
    legend.title = element_text(size = 14), # Increases legend title size
    legend.text = element_text(size = 12)   # Increases legend text size
  ) +
  labs(x = "Cell Type", y = "Gene", fill = "Relative Expression")
## Warning: Using `size` aesthetic for lines was deprecated in ggplot2 3.4.0.
## ℹ Please use `linewidth` instead.
## This warning is displayed once every 8 hours.
## Call `lifecycle::last_lifecycle_warnings()` to see where this warning was
## generated.
f4

df3 <- type_markers %>%
  filter(gene %in% gyarmati) %>%
  arrange(desc(avg_log2FC))

df3 <- rownames(df3)

df <- AverageExpression(
  object = SO4,
  features = df3,
  group.by = 'sample'
)$RNA

df
## 25 x 4 sparse Matrix of class "dgCMatrix"
##                   SO1          SO4          SO3          SO2
## Pappa2   1.631319e+27 9.502433e+35 4.599475e+50 4.357385e+66
## Frem1    5.051842e-01 7.390373e-01 2.467003e+00 4.910118e+00
## Wnt10a   4.987066e+00 1.075144e+00 9.814276e+00 3.687615e+00
## Robo2    2.266277e+16 3.841285e+08 2.292019e+13 4.007670e+20
## Hoxc4    1.163107e+00 6.831737e-01 1.048873e+00 1.148470e+00
## Irx1     6.022417e+13 7.406058e+16 2.529182e+17 1.092318e+21
## Megf9    1.206467e+02 1.525800e+02 7.966486e+03 1.738147e+03
## Irx2     5.855799e+10 8.873831e+09 1.028490e+09 1.695496e+10
## Etv1     3.699306e+00 3.019620e+01 2.293347e+01 1.632025e+02
## Bmp3     5.803240e+07 6.575880e+06 3.455761e+08 2.894297e+09
## Vegfd    2.114492e+00 3.639716e+00 4.471286e+00 6.795649e+00
## Sfrp1    2.805765e+88 3.457064e+78 5.250413e+88 1.161485e+84
## Vash2    2.151315e+00 4.729936e+00 8.024412e+00 9.960274e+01
## Spock2   2.857825e-01 1.276201e+02 8.631615e-01 7.980441e-01
## Hgfac    1.721668e+00 8.248896e-01 1.472557e+00 2.252142e+00
## Thsd4    3.658671e+01 4.429877e+02 1.772561e+02 1.065649e+02
## Adamtsl2 2.301601e+00 2.497462e+00 8.654869e+00 1.243935e+01
## Mmp14    2.742303e+00 2.985624e+00 4.180389e+00 6.459834e+00
## Hoxd11   4.322132e+00 1.988436e+00 4.259589e+00 6.610705e+00
## Pamr1    2.410947e+06 7.690718e+04 5.529267e+07 1.055369e+08
## Slit2    1.172151e+07 1.519789e+06 7.016234e+06 5.856385e+09
## Cxcl12   4.100233e+00 7.772582e-01 9.850546e-01 2.667524e+00
## Fgf9     2.161928e+04 9.677865e+04 4.320923e+04 1.409064e+07
## Fabp3    1.991354e+07 2.910558e+06 2.194586e+06 3.505956e+05
## Egfl6    1.428073e+01 1.288070e+01 1.542672e+00 6.577626e+00
df <- as.data.frame(df)

df <- t(scale(t(df)))

# convert df to tidy format

df_tidy <- df %>%
  as.data.frame() %>%
  rownames_to_column(var = "Gene") %>%
  pivot_longer(cols = -Gene, names_to = "sample", values_to = "Expression")
               

# Graph with geom_tile

df_tidy$sample <- factor(df_tidy$sample, levels = c("SO1","SO4","SO2","SO3"))

f4 <- ggplot(df_tidy, aes(x = sample, y = Gene, fill = Expression)) +
  geom_tile(color = "black", size = 0.2, width = 0.97, height = 0.90) +  # Adds thin border and spacing between tiles
  scale_fill_distiller(palette = "RdYlBu") +
  theme_minimal() +
  theme(
    axis.text = element_text(size = 12, color = "black"),
    axis.title = element_text(size = 14),
    axis.ticks = element_blank(),         # Removes tick marks
    panel.grid = element_blank(),          # Removes grid lines
    legend.position = "bottom",            # Centers the legend below the plot
    legend.title = element_text(size = 14), # Increases legend title size
    legend.text = element_text(size = 12)   # Increases legend text size
  ) +
  labs(x = "Sample", y = "Gyarmati Genes", fill = "Relative Expression")

f4

4 Grouping as Treatment groups

# 1. Create new sample groups

treatmentgroup <- df_tidy %>%
  mutate(
    sample = case_when(
      sample %in% c("SO1", "SO4") ~ "SO1_SO4",
      sample %in% c("SO2", "SO3") ~ "SO2_SO3"
    )
  ) %>%
  
  # 2. Sum (or use mean) of Expression within each group for each Gene
  group_by(Gene, sample) %>%
  summarize(Expression = mean(Expression), .groups = "drop")  # Use mean() if you prefer averaging

treatmentgroup$sample <- factor(treatmentgroup$sample, levels = c("SO1_SO4", "SO2_SO3"))
f4_grouped <- ggplot(treatmentgroup, aes(x = sample, y = Gene, fill = Expression)) +
  geom_tile(color = "black", size = 0.2, width = 0.97, height = 0.90) +
  scale_fill_distiller(palette = "RdYlBu") +
  theme_minimal() +
  theme(
    axis.text = element_text(size = 12, color = "black"),
    axis.title = element_text(size = 14),
    axis.ticks = element_blank(),
    panel.grid = element_blank(),
    legend.position = "bottom",
    legend.title = element_text(size = 14),
    legend.text = element_text(size = 12)
  ) +
  labs(x = "Control                                        Low Salt ", y = "Gyarmati Genes", fill = "Relative Expression")

f4_grouped

# HeatMap of each clusters top genes from pathway analysis

VlnPlot(SO4,split.by = "treatment","Wnt10a")